There is societal need for new compounds in our arsenal of defenses against fungal pathogens, many of which are increasingly resistant to existing therapeutics. The best possible source for new antifungal compounds with potentially novel mechanisms of action is within natural environments, particularly soils, which have the greatest diversity of microbial life. This research proposal advances the science of metagenomics, the cloning of DNA from entire microbial communities, to discover novel antibiotics and identify the best lead candidates for clinical development. Scientists at Lucigen Corporation, Auburn University, and the University of Mississippi's National Center for Natural Product Research have combined four key technological breakthroughs that result in an improved paradigm for screening metagenomic libraries for small molecules. The improvements in soil metagenomic library screening include: 1) an improved methodology for the isolation and purification of high molecular weight genomic DNA from soil microorganisms; 2) a novel broad host range shuttle vector for enhanced expression of cloned DNAs; 3) a random shear cloning method to produce very large insert sizes (>100 kb); 4) a rapid and improved screening method to identify antibiotic- producing clones within a metagenomic library. The library produced and screened in previous research resulted in the identification of 28 metagenomic clones that produce compound(s) active against methicillin-resistant Staphylococcus aureus, and one of the clones has been shown to produce novel chemical metabolites. This Phase I SBIR will build upon the success of previous research by screening these next-generation metagenomic libraries for antifungal activity. Based on previous reviewer comments, we will focus our screening efforts on metagenomic clones that contain a Type I polyketide synthase pathway, and use novel bacterial strains engineered for polyketide expression. We will characterize the antifungal agents expressed by metagenomic clones to determine the best lead candidates for clinical development. Lead candidates will have novel chemical structures, have high potency against multiple fungal pathogens, and minimal toxicity against human cell lines. Each of the different technologies necessary for the proposed research has been proven effective separately; therefore, the synthesis of these different methods has a high probability of success and also represents a significant advancement for the science of antifungal discovery.